Photodynamic therapy (PDT) has been used clinically for the treatment of head and neck cancer. The effectiveness of PDT is often strongly dependent on fluence rate. Targeted photo-immunotherapy (PIT) may reduce the adverse effects of non-targeted PDT. The in-vivo distribution of the anti-EGFR targeted conjugate Cetuximab-IRDye700DX was investigated. Vascular and tumor responses were determined with respect to fluence rate.
Intra-vital confocal microscopy of skin-fold chambers with the EGFR-overexpressing OSC-19 tumor showed peak tumor fluorescence 24 hrs after administration. Tumor to normal ratio was 3.1±1.6 (n=8). Tumor vascular responses were determined by imaging rhodamine-dextran extravasation. Two hrs after illumination (24 hr DLI, 100 J.cm-2 at 50 mW.cm-2) showed no leakage in 3 of 4 animals and stasis in 1. Normal vasculature showed mild to severe constriction of larger vessels up to 48 hrs after illumination. Subcutaneous OSC-19 tumors were transdermally illuminated with 100 J.cm-2 at 20, 50 and 150 mW.cm-2. Control tumors took 5.3±1.1 days to grow to 200%. All animals treated with 20 mW.cm-2 showed no tumor up to 90 days post treatment (n=4) compared to 1 of 4 in the 50 and 150 mW.cm-2 groups. The remaining tumors reached 200% after 17.9±5.2 and 19.5±7.4 days. Crust formation of the overlying skin was observed at low fluence rate.
Cetuximab-IRDye700DX showed significant tumor to normal ratio. Normal tissue responses like vascular effects and crust formation of the skin was observed and may be caused by conjugate still present in the circulation. The effect of targeted-PIT is strongly dependent on fluence rate.
In this study, a ratiometric quantification method is developed and applied to monitor mesotetra(hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method
employs a combination of dual-wavelength excitation and dual-wavelength detection. The excitation and
detection wavelengths were selected in close to NIR. The first excitation wavelength was used to excite the
mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be substracted.
Subsequently the difference was divided by the autofluorescence. Since the method applies division of signal
with no mTHPC fluorescence, theory suggests on linear dependency of the method on photosensitizer
concentration.
Photodynamic therapy (PDT) using protoporpyrin IX (PpIX) precursors like 5-aminolevulinic acid (ALA) or
methyl-aminolevulinate (MAL) has shown to be effective in the treatment of various skin diseases. Using ALA
we have shown in numerous studies a significantly improved efficacy by applying light fractionation with a
long dark interval. In contrast, in the hairless mouse model, the PDT efficacy using MAL is unaffected by
adopting this approach. More acute edema is found after ALA-PDT suggesting a difference in response of
endothelial cells to PDT.
To investigate the role of endothelial cells, cryo-sections of hairless mouse skin after 4 hours of topical MAL
or ALA application were stained with a fluorescent endothelial cell marker (CD31). Co-localization of this
marker with the PpIX fluorescence was performed using the spectral imaging function of the confocal
microscope. We have also used intra-vital confocal microscopy to image the PpIX fluorescence distribution in
correlation with the vasculature of live mouse skin.
Our results show PpIX fluorescence at depth in cryo-sections of mouse skin after 4 hours of topical
application. Co-localization has shown to be difficult due to the changes in tissue organization caused by the
staining procedure. As expected we found high PpIX fluorescence levels in the epidermis after both MAL and
ALA application using intra-vital microscopy. After ALA application more PpIX fluorescence was found deep in
the dermal layer of the skin than after MAL. Furthermore we detected localized fluorescence in unidentified
structures that could not be correlated to blood vessels or nerves.
Introduction: The rate of PpIX fluorescence photobleaching is routinely used as a dose metric for ALA-PDT. Diffuse
reflection spectroscopy is often used to account for variations in tissue optical properties at the photosensitizer excitation
and emission bands. It can be used to quantify changes in vascular parameters, such as blood volume fraction and
saturation, and can aid understanding of tissue response to PDT. The volume and(/or) depth over which these signals are
acquired are critical. The aim of this study is to use quantitative reflectance spectroscopy (DPS) to correct fluorescence
for changes in tissue optical properties and monitor PDT.
Materials & Methods: ALA was topically applied to hairless mice skin and the incubated spot was treated with PDT
according to fractionated illumination schemes. DPS measurements of vascular parameters and optical properties were
performed directly before and after illumination. Both the differential signal, delivery-and-collection-fiber signal and the
collection fiber signal, which all probe different measurement volumes, are analyzed.
Results & Conclusions: Analysis of DPS measurements shows that at the depth where most fluorescence originates,
there is almost no blood present. During PDT vascular parameters at this depth stay constant. In more oxygenated layers
of the tissue, the optical properties do change during PDT, suggesting that only a small part of PpIX fluorescence
originates from the interesting depths where vascular response occurs. Correcting fluorescence emission spectra for
optical changes at specific depths and not for the total of changes in a larger volume, as is usually done now, can
improve PpIX photobleaching based treatment monitoring.
PDT with ALA and MAL is established as a relatively effective treatment for non-melanoma skin cancer and premalignancies.
PDT is often repeated, because a single treatment gives poor long term results. Preclinical studies showed
that ALA-PDT applying a fractionated illumination scheme with a small first light fraction and a second larger light
fraction separated by a dark interval of two hours resulted in a significant increase in efficacy. Whereas the efficacy was
not enhanced by fractionating MAL-PDT, indicating that ALA-PDT mechanism is not the same as MAL-PDT
mechanism. The increase in efficacy using fractionated PDT was confirmed clinically. A randomized comparative
clinical study comparing fractionated ALA-PDT versus non-fractionated ALA-PDT in the treatment of superficial basal
cell carcinoma showed a significant higher response rate in the lesions treated with fractionated ALA-PDT after a
follow-up of one year ( p<0.002, log-rank test). The five year follow-up is studied at moment. So far the complete
response in the group treated with fractionated ALA-PDT seems to be only a few percentages lower compared to the one
year follow-up. Besides the gain in response rate, fractionated ALA PDT is cost effective. ALA gel is less expensive
than the commercially available MAL (Metvix) and moreover fractionated ALA-PDT takes one treatment day, instead
of two treatment days using the Metvix treatment protocol (two MAL-PDT treatments separated by one week), both
reducing direct and indirect costs and the burden to the patient.
We present an optical method based on fluorescence spectroscopy for measuring chromophore concentrations in vivo. Fluorescence differential path length spectroscopy (FPDS) determines chromophore concentration based on the fluorescence intensity corrected for absorption. The concentration of the photosensitizer m-THPC (Foscan®) was studied in vivo in normal rat liver, which is highly vascularized and therefore highly absorbing. Concentration estimates of m-THPC measured by FDPS on the liver are compared with chemical extraction. Twenty-five rats were injected with 0.3 mg/kg m-THPC. In vivo optical concentration measurements were performed on tissue 3, 24, 48, and 96 h after m-THPC administration to yield a 10-fold variation in tissue concentration. After the optical measurements, the liver was harvested for chemical extraction. FDPS showed good correlation with chemical extraction. FDPS also showed a correlation between m-THPC fluorescence and blood volume fraction at the two shortest drug-light intervals. This suggests different compartmental localization of m-THPC for different drug-light intervals that can be resolved using fluorescence spectroscopy. Differences in measured m-THPC concentration between FDPS and chemical extraction are related to the interrogation volume of each technique; ~0.2 mm3 and ~102 mm3, respectively. This indicates intra-animal variation in m-THPC distribution in the liver on the scale of the FDPS sampling volume.
Live tissue nonlinear microscopy based on multiphoton autofluorescence and second harmonic emission originating from endogenous fluorophores and noncentrosymmetric-structured proteins is rapidly gaining interest in biomedical applications. The advantage of this technique includes high imaging penetration depth and minimal phototoxic effects on tissues. Because fluorescent dyes are not used, discrimination between different components within the tissue is challenging. We have developed a nonlinear spectral imaging microscope based on a home-built multiphoton microscope, a prism spectrograph, and a high-sensitivity CCD camera for detection. The sensitivity of the microscope was optimized for autofluorescence and second harmonic imaging over a broad wavelength range. Importantly, the spectrograph lacks an entrance aperture; this improves the detection efficiency at deeper lying layers in the specimen. Application to the imaging of ex vivo and in vivo mouse skin tissues showed clear differences in spectral emission between skin tissue layers as well as biochemically different tissue components. Acceptable spectral images could be recorded up to an imaging depth of ~100 µm.
The deep-tissue penetration and submicron spatial resolution of multi-photon microscopy and the high-detection
efficiency and nanometer spectral resolution capability of a spectrograph were combined to study the intrinsic emission
of mouse skin post mortem biopsy and section, and in vivo tissue samples. The different layers of skin could be clearly
distinguished based on both their spectral signature and morphology. Auto fluorescence could be detected from both
cellular and extra cellular structures. In addition SHG from collagen and a narrowband spectral emission band related to
collagen were observed. Visualization of the spectral images in RGB color allowed us to identify tissue structures such
as epidermal cells, lipid-rich keratinocytes and intercellular structures, hair follicles, collagen, elastin, and dermal
fibroblasts. The results also showed morphological and spectral differences between the mouse skin post mortem biopsy
and in vivo samples which explained by biochemical differences, specifically of NAD(P)H. Overall, spectral imaging
provided a wealth of information not easily obtainable with present conventional multi-photon imaging methods.
We combined a homebuilt multiphoton microscope and a prism-CCD based spectrograph to develop a spectral imaging system capable of imaging deep into live tissues. The spectral images originate from the two-photon autofluorescence of the tissue and second harmonic signal from the collagen fibers. A highly penetrating near-infrared light is used to excite the endogenous fluorophores via multiphoton excitation enabling us to produce high quality images deep into the tissue. We were able to produce 100-channel (330 nm to 600 nm) autofluorescence spectral images of live skin tissues in less than 2 minutes for each xy-section. The spectral images rendered in RGB (real) colors showed green hair shafts, blue cells, and purple collagen. Analysis on the optical signal degradation with increasing depth of the collagen second-harmonic signal showed 1) exponential decay behavior of the intensity and 2) linear broadening of the spectrum. This spectral imaging system is a promising tool for both in biological applications and biomedical applications such as optical biopsy.
Interest in the development of optical technologies that have the capability of performing in situ tissue diagnosis without the need for surgical biopsy and processing has been growing. In general, optical diagnostic techniques can be classified into two categories: (1) spectroscopic diagnostics and (2) optical imaging. Spectroscopic diagnostic techniques are used to obtain an entire spectrum of a single tissue site (point-measurement method). On the other hand, optical imaging methods are aimed at recording a two- or three-dimensional image of a sample region. A third category, which combines the two modalities, is currently in an early development phase. This category, referred to as spectral imaging, has been applied to cytomics, fluorescence resonance energy transfer (FRET) analysis, histology, fluorescence microscopy and autofluorescence microscopy. In this study, we combined a multi-photon microscope with a sensitive prism-based spectrograph and employed it for intrinsic emission spectral imaging microscopy of in vivo mouse skin tissues. We show results on: (1) spectral image RGB real-color visualization; (2) tissue layer discrimination using spectral signatures; (3) depth-resolved skin tissue spectral imaging; and (4) tissue component determination by spectral (linear) unmixing.
The last two decades saw the emergence of spectroscopy and microscopic imaging as techniques for tissue diagnostics. The biochemical state of the tissue is revealed by spectroscopy, while the morphological information is visualized by microscopic imaging. Little research has been carried out to diagnose tissues based on the combination of spectroscopy and microscopic imaging. Here, we report on tissue spectroscopy and microscopic imaging employing two-photon excitation of tissue autofluorescence and second harmonic generation. We designed and constructed a prism-based spectral imaging system coupled to a two-photon microscope. Full emission spectra with a 1-7 nm spectral resolution covering 330nm to 600nm can be recorded at a maximum rate of 500 spectra per second equivalent to about 0.5 frames/min (224x224 pixels). We present results on spectral imaging of human skin sections and in-depth imaging of pig skin tissue. Different skin layers show clear differences in their intrinsic emission spectral signature that can be used for diagnosis.
We combined a non-linear microscope with a sensitive prism-based spectrograph and employed it for the imaging of the auto fluorescence of skin tissues. The system has a sub-micron spatial resolution and a spectral resolution of better than 5 nm. The spectral images contain signals arising from two-photon excited fluorescence (TPEF) of endogenous fluorophores in the skin and from second harmonic generation (SHG) produced by the collagen fibers, which have non-centrosymmetric structure. Non-linear microscopy has the potential to image deep into optically thick specimens because it uses near-infrared (NIR) laser excitation. In addition, the phototoxicity of the technique is comparatively low. Here, the technique is used for the spectral imaging of unstained skin tissue sections. We were able to image weak cellular autofluorescence as well as strong collagen SHG. The images were analyzed by spectral unmixing and the results exhibit a clear spectral signature for the different skin layers.
Neutrophils are indispensable for successful PDT. Recently it has been observed that the administration of anti-thrombocyte antiserum prevents the occlusion of the microvasculature that normally occurs upon Photofrin-based PDT. We hypothesized that this antiserium treatment would increase the therapeutic efficacy by facilitating the accumulation of neutrophils at the lesion. To study this we implanted the isologous rhabdomyosarcoma R-1 subcutaneously into the thigh of WAG/Raj rats, and treated the tumor by interstitial Photofrin-based PDT. We found that the increasing tumor doubling time after PDT under anti-thrombocyte antiserum-induced thrombocytopenia was significantly higher that in normal rats. Strikingly, the increase in tumor doubling time did not differ if thrombocytopenia was induced before or immediately after illumination. At least 1.5 times more neutrophils than normal accumulated into the PDT-treated tumors under thrombocytopenia. If the rats were rendered granulocytopenic by the administration of anti-granulocyte antiserum first followed by anti-thrombocyte antiserum post PDT, this lead to a considerable loss of the thrombocytopenia- dependent gain in the efficacy of PDT. These findings suggest that the increased accumulation of neutrophils into the tumor underlie the enhanced efficacy of PDT and may implicate that under normal clinical conditions the full granulocyte-dependent kill potential is not utilized due to the presence of activated thrombocytes that cause blood flow stasis.
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